This application is a 371 of PCT/FR00/02177 filed Sep. 13, 1999, and claims priority to foreign application FR 98/11520 filed Sep. 11, 1998.
The invention relates to the domain of vaccines and more particularly to vaccine adjuvants.
Vaccines, whether they are prophylactic or therapeutic, are intended to stimulate the immune system of the human or animal organism to which they are administered, the response of the immune system possibly being either a response of the humoral type (production of antibodies), a response of cellular type, or a combination of the 2 types of response. Conventionally, for many years, vaccination has consisted in administering to an organism a nonpathogenic version of a microorganism so as to prepare the immune system to react effectively should the organism subsequently encounter the same microorganism, in its pathogenic version. The antigen administered during the vaccination can be of various types: whole or fragmented killed microorganism, attenuated live strain of the microorganism, antigenic fractions of the microorganism or polynucleotides capable of leading to the expression by the organism of an antigenic fraction.
For a long time already, attempts have been made to increase the response of the immune system or to modify its nature, not only by acting on the antigen administered or on its method of administration, but also by adding to it immunostimulating substances or adjuvants. Since Freund""s complete adjuvant, many products have been tested, in particular inorganic salts (such as zinc chloride calcium phosphate, aluminum hydroxide or aluminum phosphate, for example), saponins, polymers, lipids or lipid fractions (Lipid A, Monophosphoryl Lipid A), etc. However, few of them have all the desired characteristics: to be good immunoadjuvants which are stable but have no risk of toxicity.
Oligonucleotides which may have immunostimulating activity are, on the other hand, known, through application WO 96/02555, these oligonucleotides possibly being administered as a vaccine adjuvant. This reference also mentions the possibility of combining with these oligonucleotides, by ionic or covalent attachment or by encapsulation, means for targeting the administration of the oligonucleotide. Such means can, in particular, consist of sterol, lipid (for example a cationic lipid, a virosome or a liposome) or an agent for specific binding to the target cell (for example a binder recognized by a receptor specific for the target cell). That application also mentions, among all the variants of use of the polynucleotides described, the possibility of administering them in conjunction with a pharmaceutically acceptable carrier vehicle. That application does not identify a vehicle as being of particular advantage, but gives an indicative list thereof and cites, in this respect, in particular solutions, solvents, dispersion media, delaying agents, emulsions and others, the use of such media for pharmaceutically active substances being mentioned as being well known in this domain.
According to the teaching of that document, the amount of oligonucleotides administered should be of sufficient amount to produce the desired biological effect.
Now, the authors of the present invention have found that, quite unexpectedly, it is possible to greatly increase the immunoadjuvant effect of an oligonucleotide without being obliged to increase the amount of oligonucleotides or the amount of antigens administered.
In order to attain this goal, a subject of the invention is an immunostimulant emulsion of the oil-in-water type, comprising at least one aqueous phase and one oily phase, characterized in that it also comprises at least one immunostimulant polynucleotide, at least one portion of which is covalently coupled to at least one lipid molecule.
For the purposes of the invention, the expression xe2x80x9cemulsion of the oil-in-water typexe2x80x9d is intended to mean a dispersion of droplets of oil in an aqueous phase which can consist of buffer such as the PBS buffer. The oily phase consists of a pharmaceutically acceptable oil which can be a mineral, animal or plant oil. Preferably, an oil which can be metabolized is used, such as squalene, esters (in particular ethyl oleate, isopropyl myristate), a plant oil (for example castor oil, sunflower oil, olive oil, etc.) or a modified plant oil (ex.: macrogol glycerides). It is possible, in particular, to obtain a satisfactory emulsion by mixing 500 mg of squalene with 10 ml of PBS buffer in a machine such as an ULTRA-TURRAX(trademark), and then microfluidizing the dispersion obtained using a microfluidizer such as the Microfluidics(trademark), which makes it possible to obtain oily particles which have a diameter of less than 200 nm.
In order to facilitate the formation of the emulsion, it is possible to also use a surfactant, in particular a surfactant which has an HLB (Hydrophilic/Lipophilic Balance) value of between 6 and 14. It is in particular possible to use a surfactant chosen from the following list of products: sorbitan esters and polysorbates, ethoxylated castor oil, which may or may not be hydrogenated, ethoxylated stearic acid, 10 EO oleyl alcohol, 20 EO cetostearyl alcohol, glycerol stearate, propylene glycol stearate, lecithins, sodium lauryl sulfate, sodium stearate, 7 EO ethoxylated glycerol cocoate, ethoxylated esters of glycerol, ethoxylated oleic acids, and mannitan oleate. Particularly good results have been obtained using TWEEN(trademark) 80.
The emulsion obtained is considered to be immunostimulant if it is capable of causing or of increasing the stimulation of the immune system, for example when it is administered together with a vaccine antigen. In this application, the emulsion is used as an immunoadjuvant.
This immunoadjuvant activity may be expressed in various ways:
make the response of the immune system to the joint administration of the antigen and of the emulsion visible, whereas the response to administration of the antigen alone was not,
increase the degree of the response of the immune system without modifying the nature thereof (for example: increase the amount of antibodies produced),
modify the nature of the response of the immune system to the administration of the antigen (for example, induce a cellular response, whereas the administration of the a alone caused only a humoral response),
induce or increase the production of cytokines, or of certain cytokines in particular.
For the purpose of the present invention, the term xe2x80x9cpolynucleotidesxe2x80x9d is understood to mean a single-stranded oligonucleotide having from 6 to 100 nucleotides, preferably from 6 to 30 nucleotides. It can be an oligoribonucleotide or an oligodeoxy-ribonucleotide. Preference is given to the use of polynucleotides comprising basic sequences with inverted symmetry, such as is the case in palindromic sequences (i.e sequences of the type ABCDEEExe2x80x2Dxe2x80x2 Cxe2x80x2 Bxe2x80x2 Axe2x80x2 in which A and Axe2x80x2, B and Bxe2x80x2, C and Cxe2x80x2, D and Dxe2x80x2, and E and Exe2x80x2 are bases which are complementary in the sense of Watson and Crick), and more particularly polynucleotides comprising at least one cytosine, guanine dinucleotide sequence in which the cytosine and guanine are not methylated. Any other polynucleotide known to be, by its very nature, immunostimulant may be suitable for the purposes of the invention. Thus, it is also possible to use the immunostimulant oligonucleotides described in Patent Application WO96/02555. Particularly good results have been obtained using a polynucleotide for which the sequence of the bases is as follows: GAGAACGCTCGACCTTCGAT.
The oligonucleotides suitable for the purposes of the invention can be in the form of phosphodiesters or, in order to be more stable, in the form of phosphorothioates or of phosphodiester/phosphorothioate hybrids. Although it is possible to use oligonucleotides originating from existing nucleic acid sources, such as genomic DNA or cDNA, preference is given to the use of synthetic oligonucleotides. Thus, it is possible to develop oligonucleotides on a solid support using the xcex2-cyanoethyl phosphoramidite method (Beaucage, S. L. and Caruthers, M. H. Tetrahedron Letters 22, 1859-1862 (1981)) for the 3xe2x80x25xe2x80x2 assembly, and then precipitation in ethanol in the presence of 0.3 M sodium acetate not adjusted for pH (0.3M final) is carried out. Next, precipitation with 4 volumes of 80% ethanol is carried out, followed by, drying before taking up the precipitate in pure water.
In the phosphorothioate-containing oligonucleotides, one of the oxygen atoms making up the phosphate group is replaced with a sulfur atom. Their synthesis can be carried out as previously described, except that the iodine/water/pyridine tetrahydrofuran solution which is used in the oxidation step required for the synthesis of the phosphodiester linkages is replaced with a TETD (tetraethylthiuram disulfide) solution which provides the sulfate ions allowing the phosphorothioate group to be produced.
It is also possible, to envisage other modifications of the phosphodiester linkages, of the bases or of the sugars, so as to modify the properties of the oligonucleotides used, and in particular so as to increase their stability.
According to the invention, at least one lipid molecule is covalently coupled to the polynucleotide. This lipid molecule is preferably a molecule of cholesterol or of a derivative of cholesterol. The coupling can be carried out by covalent attachment to one or to each end of the polynucleotide, or by insertion next to each base of at least one lipid molecule. This coupling can be carried out directly during the synthesis of the polynucleotide, using in the oligonucleotide synthesizer, a reagent such as cholesterol phosphoramidite instead of the phosphoramidite reagent conventionally used.
The antigens the effect of which it is possible to potentiate using the emulsion according to the present invention can be varied in nature; they can, in particular, be proteins, glycoproteins, glyco-conjugates, polyosides or pqlynucleotides comprising DNA fractions capable of causing the expression of molecules of interest; it can also be a mixture of various antigens. Particularly good results have been obtained with a composition comprising influenza antigens such as are present in the commercially available vaccine VAXIGRIP(trademark).
It is possible to obtain an emulsion according to the invention by carrying out the following procedure: first of all, the oil is mixed, with stirring, with the aqueous phase optionally consisting of a buffer solution into which a surfactant has been incorporated. The mixture obtained is homogenized by means, for example, of a propellor mixer, in order to produce an emulsion of the oil-in-water type. Preferably, the emulsion obtained is then treated using a microfluidizer, in order to reduce the droplets of oil to a diameter of less than 200 nm.
Then, with this emulsion being maintained with stirring, the polynucleotide to which the lipid has been coupled is simply added to it and the emulsion which is the subject of the present invention is obtained.
When this emulsion is intended to be used as an immunoadjuvant, it is mixed, with stirring, with a composition comprising the antigen the potentiation of whose effect is desired. The mixture can be advantageously prepared in a volume ratio of 1. Next, it is possible to verify the unexpected effect and in particular the synergistic effect obtained on the stimulation of the immune system by the simultaneous use of a polynucleotide coupled to at least one lipid molecule and its incorporation into an emulsion of oil-in-water type.
To this end, it is possible to carry out an immunogenicity assay on mice divided into several groups, to which mice is administered, depending on the group:
either a composition comprising only the antigen or the mixture of antigens with respect to which it is desired to test the immunostimulant effect of the emulsion according to the invention,
or a composition comprising the antigen or antigens of interest to which has been added a solution comprising only polynucleotides coupled to at least one lipid molecule,
or a composition comprising the antigen or antigens of interest to which has been added an emulsion of oil-in-water type, without polynucleotide, or with a polynucleotide lacking immunostimulant activity with respect to the antigens administered,
or a composition comprising the antigen or antigens of interest to which has been added an emulsion according to the invention.
For each of the mice immunized, it is then possible to determine the amount and nature of the antibodies produced, which makes it possible to determine the GMT (or Geometric Mean Titer of Antibodies); it is also possible to perform assays for the cytokines produced; in addition, it is possible to perform assays which enable the cellular response of the immune system to be determined.
The results obtained showed a considerable synergistic effect of the elements constituting the emulsion according to the invention.
In addition, the emulsion obtained according to the invention has increased stability with respect to emulsions of the same nature; i.e. those consisting of an identical aqueous phase and an identical oily phase, but lacking polynucleotides.